Affinity Tagging of Adipocyte Nuclei to Examine Epigenetic Controls in Obesity Project Summary/Abstract Both obesity and aging are characterized by increased adipose development in muscle and bone marrow as well as expansion of visceral adipose depots. These shifts in fat distribution are associated with decreased insulin sensitivity, increased bone loss and increased production of inflammatory cytokines. It is now well recognized that there are important regional differences in adipose tissue metabolic function and gene expression. The regulatory mechanisms involved in these regional differences are not well understood, but recent findings suggest that epigenetic changes play an important role. Furthermore, epigenetic changes are also believed to be responsible for alterations in gene expression in adipose and other tissues resulting from prenatal exposure to adverse nutritional conditions and differences among nearly genetically identically individuals in their response to obesogenic diets, caloric restriction, and exercise. I the past, epigenetic research on adipocytes has been limited by the inability to easily perform analyses of chromatin structures. Generating adipocyte populations by cell sorting or cell differentiation in culture have their limitations, not the least of which is that these manipulatios of live cells influence the precise epigenetic factors being assayed. The goal of this grant is to implement the innovative and powerful new INTACT (isolation of nuclei tagged in specific cell types) technology that will allow the rapid isolation and epigenetic analysis of fixed adipocyte nuclei from any mouse tissue. INTACT is a newly developed technology that allows the rapid isolation of cell-type specific nuclei from within an organ. It is based on the expression of a transgenic nuclear tagging protein (NTP), which uses a nuclear envelope protein to target a red fluorescent protein (mCHERRY) reporter and a biotin ligase recognition peptide (BLRP) affinity tag to the surface of cell nuclei. The BLRP peptide is biotinylated by a constitutive expressed E. coli biotin ligase (BirA). We will limit expression of the NTP fusion to adipocytes using a fragment of the adiponectin promoter (ADN5p). Affinity tagged adipocyte nuclei from different tissues of normal or environmentally compromised mice or obese mice may then be purified from mixed tissue samples on Streptavidin magnetic affinity resin. Our Specific Aims are (1) Produce and test an adipocyte-specific ADN5p:NTF reporter construct. (2) Construct an Adipocyte nuclei Biotin Labeled (ABL) transgenic mouse expressing the ADN5p:NTF gene in the homozygous ROSA26HABirA (BirA) mouse background. (3) Demonstrate the robustness and utility of the INTACT technology in mice by comparing the genome-wide methylome and transcriptome of nuclei from visceral and subcutaneous adipocytes purified on magnetic resin. This project will also serve as a demonstration of the utility of the INTACT for any cell type; thu, this proposed work will enable researchers to understand how chromatin structures and the nuclear proteome of cells in the adipocyte lineage in different tissues differ from each other and from adjacent cells. PUBLIC HEALTH RELEVANCE: Accumulating evidence indicates that epigenetic changes are responsible for differences in gene expression among different adipose depots, as well as differences in gene expression among individuals in their response to obesogenic diets, caloric restriction, and exercise; however, epigenetic studies have been limited by the difficulty in performing analyses of unstable chromatin structures. The goal of this proposal is to implement an innovative and new technology, INTACT (isolation of nuclei tagged in specific cell types), that will allow the rapid isolation and epigenetic analysis of fixed adipocyte nuclei from any mouse tissue, thus enabling us and other researchers to understand how chromatin structures and the nuclear proteome of cells in the adipocyte lineage in different tissues differ from each other and from adjacent cells. This work will greatly accelerate our understanding of the epigenome in the development of obesity and metabolic disorders.